幾丁質分解酵素基因ChiF之表現與鏈黴菌Streptomyces griseobrunneus S3對Rhizoctonia solani AG4與Pythium aphanidermatum超寄生作用之關係

The Role of ChiF Gene Expression in Relating to Mycoparasitism of Streptomyces griseobrunneus S3 on Rhizoctonia solani AG4 and Pythium aphanidermatum

楊尚書(S. S. Yang) ； 陳泰元(T. Y. Chen) ； 曾德賜(D. S. Tzeng)

《植物病理學會刊》 14卷2期 (2005/06) Pp. 147-158

https://doi.org/10.6649/PPB.200506_14(2).0007

摘要

本研究旨在瞭解屬於family 19的幾丁質分解酵素ChiF在鏈黴菌Streptomyces griseobrunneus S3對Rhizoctonia solani AG4與Pythium aphanidermatum兩種主要上傳性病原眞菌的超寄生作用中所扮演之角色。於平板培養基上行人工接種時，SGS3之孢子可在24小時內於兩供試寄主眞菌菌絲體上很快發芽並形成纏據菌絲團，以掃瞄式電了顯微鏡檢視發現SGS3之菌絲體在兩寄生真菌菌絲體上生長、纏據甚至侵入寄主體內情形，在其纏據部位下方並明顯有蝕刻性嵌入痕跡，顯示此一入侵作用明顯有細胞壁分解酵素之參與，另於部分纏據部位可見有類似吸附器構造(appressorium-like structure)之形成，唯其功能仍有待瞭解。進一步以SGS3-ChiF專一性抗血清行免疫螢光標示檢測發現，沿SGS3纏據部位均可見顯示ChiF存在之螢光反應，其中尤以在細胞壁含幾丁質的R. solani AG4菌體上可測得的螢光反應較強，而在細胞壁不含幾丁質P. aphanidermatum菌絲體上，其螢光反應相對較弱；於兩供試眞菌所測得之差異性結果，顯示SGS3 ChiF基因於幾丁質存在下有優先表現的情形，而於不含幾丁質的細胞壁上仍有弱螢光表現，應爲細胞壁分解酵素被誘導表現時多種酵素同時誘導表現之調控作用所導致。利用雙重染色技術，本研究並證實，在SGS3細胞壁分解酵素對寄主菌體尚未見有明顯的崩解效應之前，寄主細胞已明顯失去活力，此顯示於超寄生作用過程中可能尚有抗生物質的協力作用參與。本報告爲首度在鏈黴菌對病原眞菌之超寄生作用部位以免疫螢光檢測證實，有屬family 19的幾丁質分解酵素之參與作用。

關鍵字

Streptomyces griseobrunneus S3 ； 超寄生 ； ChiF蛋白 ； 幾丁質分解酵素 ； 掃瞄式電子顯微鏡 ； 免疫螢光染色 ； 活性染色

並列摘要

The explicit role of ChiF, a member of family 19 chitinase, in mycoparasitism of Streptomyces griseobrunneus S3 (SGS3) was explored using Rhizoctonia solani AG4 and Pythium aphanidermatum as targeted fungi. Upon artificial inoculation on an agar plate system, the bacterial spores appeared to germinate readily on the mycelium of both host fungi and develop well into mycelial mass on the colonized part within 24 hours. The examination by scanning electron microscopy revealed that the bacterial mycelia grew on, and in some cases penetrated the host mycelium. The mycoparasitic effect was manifested by the development of etching lesions indicating the involvement of cell wall degrading enzymes. Also worth noting was the formation of an appressorium like structure (ALS) on host mycelium, although its role in the mycelial penetration remained to be determined. The involvement of ChiF in the observed mycoparasitism was well illustrated by an immuno-fluorescent microscopy where that SGS3-ChiF specific polyclonal antibody was applied. The fluorescent signal indicating the expression of ChiF gene was detected along the contact interface of SGS3 on the host mycelium. The detected signal was strong especially among those colonizing on R. solani; the fluorescence detected from that on P. aphanidermatum was comparatively low. The differential response observed implicated a preferential expression of the gene on the fungal cell wall with chitin as a major constituent. The low expression detected on the non-chitin cell wall of P. aphanidermatum further implicated the functioning of co-regulation of cohorts of cell wall degrading enzymes in the applied mycoparasite. By double staining of fluorescent diacetate and propidium iodide, it was shown further a complication of antibiotic in the observed mycoparasitism since the colonized cells of both targeted fungi lost their viability before the dismantling effect of the bacteria-secreted cell wall degrading enzymes became apparent. This is a first report of in situ detection of family 19 chitinase gene expression illustrating the involvement of ChiF chitinase in Streptomyces mycoparasitism.

並列關鍵字

Streptomyces griseobrunneus S3 ； mycoparasitism ； ChiF potein ； chitinase ； scanning electron microscopy SEM ； immuno-fluorescent staining ； viability staining